Immunization using plasmid DNA-based immunogenic compositions is a powerful tool that is useful for developing approaches to prevent or treat infectious diseases or in the treatment of ongoing disease processes. Plasmid DNA immunization has been extensively tested in animal models where it has been found to be effective in inducing both cellular and humoral immune responses against a wide variety of infectious agents and tumor antigens. See Donnelly J J, et al., Ann. Rev. Immunol.; 15: 617-48 (1997); Iwasaki A, et al., J Immunol 158 (10): 4591-601 (1997); Wayne, C. L. and Bennett M., Crit. Rev. Immunol., 18: 449-484 (1998).
An important advantage of plasmid DNA immunization is that genes can be cloned, modified and positioned into a potential plasmid DNA expression vector in such a way as to allow for relevant post-transcriptional modifications, expression levels, appropriate intracellular trafficking and antigen presentation. Plasmid DNA vectors useful for DNA immunization are similar to those employed for delivery of reporter or therapeutic genes. Plasmid DNA-based immunization uses the subject's cellular machinery to generate the foreign protein and stimulates the subject's immune system to mount an immune response to the protein antigen. Such plasmid DNA vectors generally contain eukaryotic transcriptional regulatory elements that are strong viral promoter/enhancer elements to direct high levels of gene expression in a wide host cell range and a polyadenylation sequence to ensure appropriate termination of the expressed mRNA. While, viral regulatory elements are advantageous for use in plasmid DNA vectors, the use of unmodified viral vectors to express the relevant genes may raise safety and technical issues not encountered with plasmid DNA.
Current plasmid DNA designs, however, limit the expression of multiple genes from one vector backbone in a single target cell. Therefore, to transfer and express multiple genes, co-transfection of the target cells with separate plasmids is required. When cells must be co-transfected with multiple plasmids, it is difficult to achieve optimal expression of all encoded genes, especially when the plasmid is being used in vivo. Previous attempts to overcome these limitations and express two or more genes include the use of the following: viral vectors, multiple alternatively spliced transcripts from proviral DNA, fusion of genes, bicistronic vectors containing IRES sequences (Internal ribosome entry site) from viruses and dual expression plasmids. See Conry R. M. et al., Gene Therapy. 3(1):67-74, (1996); Chen T T. et al., Journal of Immunology. 153(10):4775-87, (1994); Ayyavoo V. et al., AIDS. 14(1):1-9, (2000); Amara R. R. et al., Vaccine. 20(15):1949-55, (2002); Singh G, et al., Vaccine 20: 1400-1411 (2002).
None of the existing plasmid designs have solved the problem of providing a DNA plasmid suitable for expressing more than two independent open reading frames in human immunogenic compositions. In the case of bicistronic vectors, in many instances, only the first gene transcribed upstream of the IRES is expressed strongly from either a plasmid or a retroviral vector. See Sugimoto Y., et al., Hum. Gen. Ther. 6: 905-915 (1995); Mizoguchi H, et al., Mol. Ther. 1:376-382 (2000). Dual expression cassettes on the other hand have performed better. For example, it was found that co-delivery of cDNA for B7-1 and human carcinoembryonic antigen (CEA) with a single plasmid having two independent cassettes resulted in far superior immune responses, when compared to separate plasmids. See Conry R. M. et al., Gene Therapy. 3(1):67-74, (1996). However, in this case the two independent cassettes involved both consisted of homologous HCMV promoter and bovine growth hormone (BGH) poly-adenylation sequences. The presence of homologous sequences within a plasmid renders that plasmid unsuitable for use in DNA immunogenic compositions, because the presence of homologous sequences within the plasmid backbone increases the possibility of recombination between the repeated sequences and results in vector instability.
Another constraint one confronts when designing a plasmid DNA vector for use in a human immunogenic composition involves size and organization of the plasmid. As transcriptional units are added to a plasmid, interference between transcriptional units can arise, for example in the form of steric hindrance. The cell's RNA transcription complex must be able to bind to the multiple sites on a polytranscriptional unit plasmid, uncoil the DNA and effectively transcribe the genes. Simply making the plasmid bigger is not necessarily the best solution for several reasons including plasmid instability, difficulty in plasmid manufacture and, most importantly, dosing considerations. To design an improved plasmid DNA multiple transcriptional unit vector, one must consider placement of genes, spacing and direction of transcription of open reading frames, level of expression, ease of manufacture, safety and the ultimate dose of the vector necessary to immunize the subject.
Therefore, there remains a need for innovative plasmid DNA, non-viral vector designs for use in expressing multiple proteins from complex pathogens like HIV, where a broad immune response to many proteins is required. In addition, a need exists for polyvalent DNA-based immunogenic compositions that can direct expression of high levels of multiple HIV genes within a single cell.